Which hormone is produced by the pancreatic islet cells and is responsible for lowering the concentration of glucose in the blood?

Organization of the Adult Pancreas

Islets of Langerhans are distributed throughout the adult organ and are supported by a mass of branching exocrine tissue. Islets vary considerable in size, but a typical islet is around 50–200 μM in diameter. In mouse islets, β cells are found to be clustered together in the core of each islet, surrounded by peripheral hormone secreting cells, most abundantly α cells. In contrast in the human pancreas, endocrine cell types do not exhibit such distinct architecture but appear intermixed within the whole islet. It has been suggested that the predominant homotypic β–β cell interactions in mice compared to mostly heterotypic interactions of β–non-β cells in humans could be one reason for observed species specific differences. However, in both species, δ cells have long processes that invade the islet and make contact with several α and β cells.

While islets of Langerhans make up only an estimated 1% to 2% of the total pancreas, they receive up to 20% of its blood supply. The rich vascular blood supply of the pancreas originates from the splenic artery allowing islets to be readily exposed to systemic blood glucose concentrations. Interestingly, a recent study suggests that local blood flow in islets is highly regulated by perciytes that control the diameter of islet microvasculature in a dynamic fashion. Under diabetic conditions, perciytes are largely lost and fine tuning of islet function by this mechansim is impaired, suggesting a contribution to disease pathology.

Pancreata from adult mice and humans also differ in terms of innervation. Mouse islets have extensive networks of branching nerves which make direct contact with endocrine cells while human islets have been found to be sparsely innervated by comparison—with the nerves that do exist, making contact with smooth muscle cells found on blood vessels rather than endocrine cells. This supports the emegering hypothesis that in the human, regulation of islet function is more likely to be dependent on dynamic control of blood flow than direct signals from the autonomic nervous system.

Introduction

Islets of Langerhans are three-dimensional clusters of approximately 1000 cells that constitute the endocrine portion of the pancreas, and each islet is around 50–500 μm in diameter. The most abundant islet cell type in all species is the insulin-secreting β-cell, although there is some variation in the proportion of β-cells between species, with estimates that mouse islets comprise 80–90% β-cells, while in human islets the β-cells contribute 60–70% to the islet mass.1 There are many similarities in the functional characteristics of rodent and human islets, and since islets isolated from cadaver organ donors are not widely available for research many studies over the past 50 years have made use of mouse and rats islets. Islets from these rodents are similar in size to human islets. However, a mouse pancreas is considerably less than 0.1% of the volume of a human pancreas, so while 250,000–500,000 islets can be obtained from a human pancreas the yield from a mouse pancreas is only 200–250 islets. In addition, islet isolation from all species by collagenase digestion of the exocrine pancreas and purification by handpicking or density gradients is time consuming. Furthermore, while some primary cells, such as those derived from smooth muscle, proliferate in culture to produce additional cells for experimental use, islet cells do not readily proliferate. Therefore, considerable effort has been expended since the 1970s to generate insulin-secreting cells that proliferate in culture and show functional characteristics of primary β-cells. These immortalized insulin-secreting cell lines, which can be maintained in continuous cultures, have been developed by a number of methodologies and they are the subject of this chapter.

Etiology

The islets of Langerhans are the pancreatic endocrine centers.62 These groups of cells are found throughout the pancreas but comprise only about 2% of the total pancreatic tissue.62 Normal pancreatic islets contain four cell types that each secrete a different peptide: alpha cells secrete glucagon, beta cells secrete insulin, delta cells secrete somatostatin, and P (F) cells secrete pancreatic polypeptide.41 Neuroendocrine tumors of the pancreas are neoplasms arising from the islets of Langerhans and are called islet cell tumors.15 These tumors are further classified by the type of peptide they secrete. Tumors secreting biologically active peptides that result in clinical symptoms are called functional islet cell tumors.15 The most frequently reported functional tumor in small animals and specifically in ferrets is the insulin-secreting beta-cell tumor, or insulinoma.18,27,31,62,84 In one study, 94% of beta-cell tumors in ferrets were reported to be functional.57 In fact, islet cell tumors are reported to be the most common endocrine neoplasm seen in ferrets, with an incidence of 22%57 of all reported neoplasms in a 1998 retrospective study and an incidence of 25% of all reported neoplasms in a 2003 retrospective study.57,113

Overview

The islets of Langerhans are endocrine functional units of the pancreas. Histologically, four cell types can be identified: the α or A cells that secrete glucagon; the β or B cells that secrete insulin; the δ or D cells that secrete somatostatin; and the PP cells that secrete pancreatic polypeptide.

Exocrine pancreatic tumours (adenocarcinomas) are more frequent than endocrine (islet cell) tumours. One single case report on a pancreatic adenoma of pancreatic islet tissue in a 12-year-old Shetland pony mare has been published.48 The pony had eight seizures over a 2-month period. Plasma glucose levels during three of these seizures were markedly depressed and serum insulin levels were elevated during two of the seizures. Gross and microscopic pathological evaluation of the pancreas revealed a single adenoma of pancreatic islet cell origin and hyperplasia of islet cells, predominantly beta cells.48

Islets of Langerhans: The Endocrine Pancreas

‘Where are the islets of Langerhans?’ is the only question relating to histology likely to be heard in a general knowledge quiz. The answer is, of course, ‘in the pancreas’. Langerhans first described these micro-organs in the rabbit in 1869 (Baskin, 2015), but they were soon identified in mammals and other vertebrates. Similar tissue occurs as endocrine follicles in the wall of the gut in cyclostomes (lampreys and hagfish), and as discrete organs in some teleost fish (Romer, 1970). The major products of the islets are insulin and glucagon, though a number of other products have been identified (see Fig. 16.5).

Etiopathogenesis

The islets of Langerhans contain four cell types that each secrete a different peptide: alpha cells secrete glucagon, beta cells secrete insulin, delta cells secrete somatostatin, and P (F) cells secrete pancreatic polypeptide.28 In healthy individuals, the ratio between the blood insulin and glucose concentrations remains constant because of control by the beta cells of the pancreatic islets.22 Insulin secretion increases when blood glucose concentration are high, while insulin secretion is inhibited when blood glucose is low.22 When neoplastic beta cells are present, they synthesize and release insulin autonomously despite hypoglycemia.22

Although a genetic component has been suggested in the development of insulinoma in ferrets, the exact cause is unknown.11 Based on the natural carnivorous diet of mustelids, a diet high in carbohydrates has been suggested as a contributing factor to the development of these tumors.23 On a dry-matter basis, a diet high in protein (42%–55%) and fat (18%–30% ) and low in carbohydrates (8%–15%) and fiber (1% to 3%) has been advised, because this may help to reduce the incidence of the disease.23 A proposed alternative is feeding entire prey animals or a canned food diet with no carbohydrates. However, currently no studies have been done to support these suggestions.

Microvasculature

The islets of Langerhans have a glomerular-like capillary network with a direct arteriolar blood supply. One to three arterioles penetrate each islet through discontinuities of the non-beta cell mantle and enter directly into the beta cell core, where each branches into a number of fenestrated capillaries. These capillaries follow a tortuous path, passing first through the beta cell core and then through the non-beta cell mantle. Often, a capillary will pass along the inside of the mantle before penetrating it to leave the islet. The pattern of microvasculature varies with islet size. In small islets (<160 μm in diameter), the efferent capillaries pass through exocrine tissue for 50 to 100 μm before coalescing into collecting venules. Large islets (>200 μm in diameter) are selectively located near the larger ducts and blood vessels. Their efferent vessels coalesce within the islet capsule, thus they probably have little effect on surrounding exocrine tissue. However, the vascular pattern of the small islets and their abundance would lead to an effective insuloacinar portal system.

The blood flow to the islets has been found to be disproportionately large (10–20% of the pancreatic blood flow) for the 1–2% of pancreatic volume. High concentrations of glucose have been shown to enhance pancreatic blood flow and to preferentially increase islet blood flow. Lymphatic vessels, although common in the pancreas, are not found within the islets.

Distension and Digestion

The islets of Langerhans remain surrounded by exocrine and connective tissues within the pancreas, which remain a limiting factor in the success of islet isolation and subsequent transplantation. The digestion process is a delicate process that must consider various parameters such as the anatomy of the pancreas, method of introducing the enzymes, and the types of enzymes. Different enzymes, such as collagenase and protease, has variability in enzyme activity and concentration between enzyme batches that hinders the consistency and reproducibility in islet isolation procedures. These factors affect the functional, ultrastructural, and immunological characteristics of the islets.

After the sterilization and trimming process, the pancreas is cut in half and the duct on both halves is cannulated. The islets are freed from the structure as the digestive enzyme is distended through the main pancreatic duct; the exocrine and connective tissues are digested as the ducts branch off into smaller ducts, penetrating further into the pancreas. Distension must be done evenly to ensure the release of maximum number of islets, and this can be done either manually by hand or mechanically with a perfusion device where pressure is controlled. It has been demonstrated that although using the perfusion machine resulted in greater islet yield postpurification, the in vitro islet function was not significantly different between hand-pumping and use of a perfusion device.

After the distension, the pancreas is then finely chopped into ∼ 2 cm pieces and placed into a metal dissociation chamber (Ricordi Chamber, which was introduced by Ricordi and colleagues in 1988) containing steel ball bearings. The islets are separated from exocrine and connective tissues in a closed-circuit system where the media and enzymes are circulated. In order to for enzymes to function optimally, the temperature of the fluids used is brought up to 37°C. Throughout the circulation, frequent sampling is used to determine the completion of digestion.

II Morphology and Hormones of the Islets of Langerhans

The islets of Langerhans comprise approximately 1-2% of the weight of the pancreas and are more concentrated in the tail than in the head or body of the pancreas. Islets can be isolated and separated completely from the exocrine pancreas by the collagenase technique, and the isolated islets will form, store, and release insulin in vitro. This procedure has made it feasible to accomplish detailed morphological and biochemical studies on each of these basic processes under controlled conditions. With scanning electron microscopy the isolated islets appear as round or ovoid structures, and the three-dimensional appearance of the capillary network can be visualized after the vascular injection of a plastic with subsequent digestion of the parenchyma. The islet cells are apparently interposed between and around this network of capillaries. With specific staining procedures, alpha, beta, and delta cells can be identified by light microscopy in the islets of the human pancreas. With the fluorescent antibody technique, insulin can be demonstrated in the beta cell and glucagon in the alpha cell. Dr. Greider, in our department, has demonstrated gastrin-containing cells in the islets of the normal human pancreas using the fluorescent antibody procedure. Presumably these gastrin-containing cells in the islets are delta cells; however, definitive proof of this relationship must await electron microscopic immunochemical identification using peroxidase-labeled antibodies to gastrin. These three cell types in the human islet can be identified with electron microscopy based upon ultrastructural differences in their secretory granules: Beta granules are round or rectangular with a crystalline structure; alpha granules are round with a central dense core; delta granules are round, less dense than alpha granules and have an amorphous appearance. The individual islet cells are surrounded by a continuous plasma membrane with scattered desmosomes attaching them to the plasma membrane of neighboring islet cells. The islet capillaries are lined with a fenestrated-type of endothelium, and two basement membranes separate the capillaries from the islet cells (Fig. 1). Frequently nerve fibers can be observed between the two basement membranes as well as immediately adjacent to the plasma membrane of islet cells. Presumably, these fibers are innervating the islet cells. Other types of islet cells have also been identified with light and electron microscopy, such as the X cell in the uncinate process of the dog pancreas. Future studies will undoubtedly identify additional hormones in the islets that will be localized to these other types of islet cells.

2.3 Pathophysiology

The islets of Langerhans in the rat produce several polypeptide hormones and amines, the most abundant of which are insulin and glucagon. Other hormones in the rat pancreatic islets are somatostatin, PP, substance P, and ghrelin. The various islet products are involved in regulation of multiple metabolic activities. Insulin accounts for approximately 85% of the hormone production of the endocrine pancreas, and has the primary function of facilitating entry of glucose through cell membranes. In addition, it influences glucose utilization by controlling gluconeogenesis in liver, muscle, and adipose tissue. By these dual mechanisms, insulin maintains blood glucose levels within the appropriate physiological range. Insulin also has metabolic effects less directly concerned with glucose utilization. These include facilitation of amino acid and fatty acid transport across cell membranes, stimulation of anabolic pathways, and release of growth hormone and ACTH from the pituitary gland. Glucagon promotes glucose mobilization by stimulating hepatic glycogenolysis and gluconeogenesis from amino acids and fatty acids. Somatostatin, named for its action in suppressing secretion of growth hormone, also inhibits gastrointestinal motility, splanchnic blood flow, gastric acid secretion, pancreatic exocrine secretion, secretion of insulin and glucagon, and absorption of triglycerides. Control of the release of islet cell hormones is influenced by four major mechanisms: (i) blood levels of certain nutrients including glucose, fatty acids, and amino acids; (ii) postprandial secretion of incretin hormones, such as glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP) from enteric endocrine cells that stimulate glucose-dependent insulin secretion; (iii) activity of the autonomic nervous system (parasympathetic stimulation favors secretion of both insulin and glucagon, whereas sympathetic activity inhibits insulin release and promotes glucagon secretion); and (iv) paracrine activity of islet hormones on neighboring cells. By this latter mechanism, insulin inhibits glucagon release, glucagon stimulates release of insulin, and somatostatin inhibits release of both insulin and glucagon. Insulin secretion by β cells declines with age. When maximally stimulated by glucose, the insulin secretion per unit of endocrine tissue of 12-month-old rats is 25–33% of that of 2-month-old rats. This is true whether the animals are maintained on a standard diet fed ad libitum or on a calorie-restricted diet. The diminished insulin responsiveness may be partly attributable to increased somatostatin. However, the mass of the endocrine pancreas is three- to fourfold greater in the mature ad libitum-fed rat than in 2-month-old rats and therefore, the total insulin secretion of the pancreas is nearly the same. The greater islet cell mass is due to larger number and volume density of β cells, whereas α- and δ-cell populations remain approximately the same. In rats fed a calorie-restricted diet, the endocrine cell mass is not as great as that in ad libitum-fed rats.

DM is the clinical condition where there is failure of the control of blood glucose with the development of hyperglycemia and hyperglucosuria. There are many serious sequelae to DM, including increased predisposition to infections, cataract formation, hepatic lipidosis and cirrhosis, atherosclerosis, microangiopathy (such as in the retina, glomerulus, and skin), peripheral neuropathy, glomerulosclerosis, and renal failure. Type I DM (T1DM) is a chronic autoimmune disease that results in a primary lack of β cells and decreased insulin production and secretion. T1DM is usually seen in young animals or children and is due to a genetic lack or inflammatory destruction of β cells. Type 2 DM (T2DM) is the most common form of DM in humans. T2DM is a heterogeneous group of disorders and is associated with obesity, physical inactivity, and genetic predisposition. It is initially due to impaired insulin action from reduced sensitivity of insulin receptors to insulin. Insulin levels may be initially elevated but then are reduced when there is failure of the β cells with prolonged T2DM. In animals, T2DM occurs in obese mice, rats, cats, rabbits, horses, and pigs. Interestingly, obese dogs are resistant to development of T2DM. Some strains of rats are predisposed to developing T2DM with obesity. Rat models of T2DM include the Zucker diabetic fatty (ZDF) rat, sand rat, high fat diet/streptozotocin treatment, and complete or partial pancreatectomy, among others.